Overlapping functionality of the Pht proteins in zinc homeostasis of streptococcus pneumoniae

Streptococcus pneumoniae is a globally significant pathogen that causes a range of diseases, including pneumonia, sepsis, meningitis, and otitis media. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including transition metal ions such as zinc. The pneumococcus employs a number of surface proteins to achieve this, among which are four highly similar polyhistidine triad (Pht) proteins. It has previously been established that these proteins collectively aid in the delivery of zinc to the ABC transporter substrate-binding protein AdcAII. Here we have investigated the contribution of each individual Pht protein to pneumococcal zinc homeostasis by analyzing mutant strains expressing only one of the four pht genes. Under conditions of low zinc availability, each of these mutants showed superior growth and zinc accumulation profiles relative to a mutant strain lacking all four genes, indicating that any of the four Pht proteins are able to facilitate delivery of zinc to AdcAII. However, optimal growth and zinc accumulation in vitro and pneumococcal survival and proliferation in vivo required production of all four Pht proteins, indicating that, despite their overlapping functionality, the proteins are not dispensable without incurring a fitness cost. We also show that surface-attached forms of the Pht proteins are required for zinc recruitment and that they do not contribute to defense against extracellular zinc stress

Heterogeneous nucleation is required for crystallization of the ZnuA domain of pneumococcal AdcA

Zn2+ is an essential nutrient for all known forms of life. In the major human pathogen Streptococcus pneumoniae, the acquisition of Zn2+ is facilitated by two Zn2+-specific solute-binding proteins: AdcA and AdcAII. To date, there has been a paucity of structural information on AdcA, which has hindered a deeper understanding of the mechanism underlying pneumococcal Zn2+ acquisition. Native AdcA consists of two domains: an N-terminal ZnuA domain and a C-terminal ZinT domain. In this study, the ZnuA domain of AdcA was crystallized. The initial crystals of the ZnuA-domain protein were obtained using dried seaweed as a heterogeneous nucleating agent. No crystals were obtained in the absence of the heterogeneous nucleating agent. These initial crystals were subsequently used as seeds to produce diffraction-quality crystals. The crystals diffracted to 2.03 angstrom resolution and had the symmetry of space group P1. This study demonstrates the utility of heterogeneous nucleation. The solution of the crystal structures will lead to further understanding of Zn2+ acquisition by S. pneumoniae

The first histidine triad motif of phtd is critical for zinc homeostasis in Streptococcus pneumoniae

Streptococcus pneumoniae is the world's foremost human pathogen. Acquisition of the first row transition metal ion zinc is essential for pneumococcal colonization and disease. Zinc is acquired via the ATP-binding cassette transporter AdcCB and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that AdcAII is reliant upon the polyhistidine triad (Pht) proteins to aid in zinc recruitment. Pht proteins generally contain five histidine (His) triad motifs that are believed to facilitate zinc binding and therefore play a significant role in pneumococcal metal ion homeostasis. However, the importance and potential redundancy of these motifs have not been addressed. We examined the effects of mutating each of the five His triad motifs of PhtD. The combination of in vitro growth assays, active zinc uptake, and PhtD expression studies show that the His triad closest to the protein's amino terminus is the most important for zinc acquisition. Intriguingly, in vivo competitive infection studies investigating the amino- and carboxyl-terminal His triad mutants indicate that the motifs have similar importance in colonization. Collectively, our new insights into the contributions of the individual His triad motifs of PhtD, and by extension the other Pht proteins, highlight the crucial role of the first His triad site in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence

Selective ferroptosis vulnerability due to familial Alzheimer's disease presenilin mutations

Mutations in presenilin 1 and 2 (PS1 and PS2) cause autosomal dominant familial Alzheimer's disease (FAD). Ferroptosis has been implicated as a mechanism of neurodegeneration in AD since neocortical iron burden predicts Alzheimer's disease (AD) progression. We found that loss of the presenilins dramatically sensitizes multiple cell types to ferroptosis, but not apoptosis. FAD causal mutations of presenilins similarly sensitizes cells to ferroptosis. The presenilins promote the expression of GPX4, the selenoprotein checkpoint enzyme that blocks ferroptosis by quenching the membrane propagation of lethal hydroperoxyl radicals. Presenilin gamma-secretase activity cleaves Notch-1 to signal LRP8 expression, which then controls GPX4 expression by regulating the supply of selenium into the cell since LRP8 is the uptake receptor for selenoprotein P. Selenium uptake is thus disrupted by presenilin FAD mutations, suppressing GPX4 expression. Therefore, presenilin mutations may promote neurodegeneration by derepressing ferroptosis, which has implications for disease-modifying therapeutics.Peer reviewe

The structural basis of bacterial manganese import

肺炎球菌が細胞内にマンガンイオンを取り込むしくみ --膜輸送体PsaBCの立体構造の解明--. 京都大学プレスリリース. 2021-09-15.Metal ions are essential for all forms of life. In prokaryotes, ATP-binding cassette (ABC) permeases serve as the primary import pathway for many micronutrients including the first-row transition metal manganese. However, the structural features of ionic metal transporting ABC permeases have remained undefined. Here, we present the crystal structure of the manganese transporter PsaBC from Streptococcus pneumoniae in an open-inward conformation. The type II transporter has a tightly closed transmembrane channel due to “extracellular gating” residues that prevent water permeation or ion reflux. Below these residues, the channel contains a hitherto unreported metal coordination site, which is essential for manganese translocation. Mutagenesis of the extracellular gate perturbs manganese uptake, while coordination site mutagenesis abolishes import. These structural features are highly conserved in metal-specific ABC transporters and are represented throughout the kingdoms of life. Collectively, our results define the structure of PsaBC and reveal the features required for divalent cation transport

EIN ALTKROATISCHES GRABERFELD AUF DEM BERGE SPAS BEI KNIN

Autor objavljuje rezultate istraživanja starohrvatske nekropole na brdu Spasu kod Knina (otkriveno ukupno 228 grobova). Na temelju prikupljenih podataka tijekom višegodišnjih arheoloških istraživanja i sprovedene analize grobne arhitekture, pogrebnih običaja i grobnih nalaza utvrđeno je vrijeme ukopavanja na toj nekropoli i uputilo na mogući prostorni smještaj naselja kojemu je to groblje pripadalo. Završna razmatranja objelodanjenih grobnih nalaza otkrivaju niz novih spoznaja o gospoda.stvenim mogućnostima stanovništva, njihov kulturni i duhovni život u 9, 10. i 11. stoljeću. Osim starohrvatskih grobova na Spasu su otkopani i temeljni ostaci arhitekture, za koje se utvrdilo da pripadaju rimskom naselju, vjerojatno ostacima stare Ninie, odnosno Curcuma. Sve to popraćeno je obilnom dokumentacijskom građom i katalogom grobova.Westlich der Stadt Knin erstreckt sich der Berg Spas. Auf seinem siidlichen Teil erhebt sich die mittelalterliche Festung Knin, und an den nord lichen Teil schlieBt sich das Hochplateau des Berges Spas an, das im horizontalen Querschnitt cinE\u27 birnentormige Form hat. Spas ist anfangs des 18. lahrhunderts durch einen kiinstlichen Durchstich von der Festung getrennt worden. Im nordlichen, breitesten Teil des Plateaus wurde ein altkroatisches Graberfeld entdeckt, mit insgesamt 228 Grabern. Das erforsci1te Gebiet umfaBt etwa 1200 m2 Oberflache. Es wurden neben Grabern auch Fundamente von Bauwerken aus vorktoatischer Zeit gefunden, was bezeugt, daB es sich hier um eine komplexe archaologische Fundstatte handelt. Im ersten Teil seiner Abhandlung beschreibt. der Verfasser die Lage des Fundortes, seine strategische Bedeutung, und den geschichtlichen Ablauf der Forschungen an diesem Ort. AuBerdem erwahnt er unter anderen bedeutenden Funden auch die Entdeckung von Fundamenten von Bauwerken , fUr die er aufgrund von Fragmenten steinernen Skulpturen, Ziegeln und Fragmenten von KeramikgefaBen festelIen konnte, daB sie einer romisehen Ansiedlung angehorten. Durch Suchgrabung wurde festgestelIt, daB die Reste dieser Bauwerke sich auch auf den iibrigen Teil des Plateaus erstrecken, der bis jetzt nicht erforscht worden ist. Diese Entdeckungen, zusammen mit einem groBen, schon friiher untersuchten Graberfeld am ostlichen FuBe dieses Berges, genannt »Greblje«, sind ein starkes Argumentfi.ir die Annahme jener Forscher, die der Meinung sind, daB die alte, antike Stadtsiedlung Ninia, die von der Schriftstellern der Antike erwahnt wird , sich an der SteIle der heutigen Stadt Knin befunden hatte, beziehungsweise an der Stelle ihrer Festung und der Wallburg Spas. Im siidostlichen Teil des ausgegrabenen Graberfeldes hat man ziemlich groBe Schuttablagerungen gefunden, unter denen sich auch Stiicke von Mortel mit Farbresten befanden. An dieser Stelle und in ihrer unmittelbaren Nahe wurden einige steinerne Fragmente mit vorromanischer F1echtwerkplastik gefunden , die zu Kircheneinrichtungsgegenstanden aus dem 9. oder 10. lh. gehorten. Die erwahnten Funde weisen mjt Sicherheit darauf hin, daB sich an eben dieser Stelle, wo man den Schutt gefunden hat, eine altkroatische Kirche befunden hatte, der die gefundenen Fragmente angehorten. Da sich von diesem Kirchenbau auch nicht der allergeringste Rest einer Mauer erhalten hat, kann man nur schlieBen, daB die Kirche bis zu den Grundmauern zerstort worden war, und daB ihre Steine als Baurnaterial fUr die Mauern der nahegelegenen Festung verwendet wurden. Der zwei te Teil dieser Abhandlung enthalt den Katalog der Grabarchitektur und ihres lnventars. Hier findet man kurz gefaBt die wichtigsten Angaben i.iber jedes cinzeIne Grab und die osteologischen Funde, sowie eine genaue Beschreibung der Grabbeigaben. Der dritte Teil behandelt die allgemeinen Merkmale des Graberfeldes. Wegen der ungi.instigen Bodenstruktur, sowie der Fundamente der Bauwerke, besitzt das Graberfeld nicht die Merkmale von Reihengrabern, Die Graber sind sehr verschiedenartig orientiert, und liegen in allen Richtungen; der groBte Teil der Graber war trotzdem richtjg orientiert, in der Richtung Osten-Westen (103 Graber) . In den meisten Grabern befand sich nur ein Verstorbener, manchmal jedoch auch mehrere. Die gefundenen osteologischen Reste bestatigen, daB das Graberfeld einer Siedlung zugehorig; mit einer gleich groBen Anzahl von bestatteten Mannern , Frauern und Kindern. Die Graber waren meistens aus behauener Rauhwacke , aus unregelmaBigen Steinplatten oder aus Bruchsteinen gebaut. Die behauene Rauhwacke ftir die Graber stammte wahrscheinlich von den eingesttirzten Bauwerken in der nahen Umgebung. Weiterhin analysiert der Verfasser die Grabfunde, meistens Schmuck, und zwar Ohrringe und Ringe. Indem er die typologischen Besonderheiten der einzelnen Gegenstande genau beurteilt, und sie mit analogem Material aus den tibrigen altkroatischen und anderen Graberfeldern vergleicht, kommt er zu Schltissen ber die Herkunft und\u27 Datierung der Funde. Mehr Aufmerksamkeit schenkt der Verfasser den Ohrringen vom Typ mit drei Beeren und zwar deshalb, weil in letzter Zeit versucht wurde, fast alle entwickelteren Varianten dieses Typs, die in Grabern auf dem Gebiet des altkroatisches Staates gefunden worden waren, von allen tibrigen Ohrringen die ebenfalls auf diesem Gebiet gefunden wurden, zu trennen. [ndem er auf das Problem der Datierung und Herkunft der Ohrringe mit drei Beeren eingeht, versuchte der Verfasser aufgrund einer Reihe sicherer Tatsachen und Hinweise darauf hinzuweisen, daB dieser Typ von Ohrringen, beziehungsweise die starker entwickelten Varianten , nicht von den tibrigen Typenvarianten altkroatischer Ohrringe isoliert werden dtirfen, die auf dem Gebiet des dalmatinischen Kroatiens gefunden worden waren , und die meistens in die Zeit vom Beginn des 9. bis zum Ende des 11. lahrhunderts datiert werden. Er machte ebenfalls darauf aufmerksam, daB samtliche Typen von altkroatischen Ohrringen, und so auch die entwickelteren Varianten von Ohrringen mit drei Beeren, die aus Grabern des Mutterlandes stammen, aus einheimischen Goldschmiedewerkstatten stammen , und daB sie als Ganzes eine altkroatische Kulturgruppe bilden. AuBer seinen Vermutungen tiber die Lage und GroBe der Ansiedlung, zu der das Graberfeld auf dem Berge Spas gehorte, ist der Verfasser im Ganzen der Meinun~, daB das Graberfeld auf Spas dem Zeitraum des frtihfeudalen kroatischen Staates angehorte, d. h., daB das Graberfeld sei t dem Anfang des 9. Jahrhunderts bestanden hatte und kontinuierlich bis zum Ende des ll. Jahrhunderts fortdauerte

Characterizing the conformational dynamics of metal-free PsaA using molecular dynamics simulations and electron paramagnetic resonance spectroscopy

Prokaryotic metal-ion receptor proteins, or solute-binding proteins, facilitate the acquisition of metal ions from the extracellular environment. Pneumococcal surface antigen A (PsaA) is the primary Mn2+-recruiting protein of the human pathogen Streptococcus pneumoniae and is essential for its in vivo colonization and virulence. The recently reported high-resolution structures of metal- free and metal-bound PsaA have provided the first insights into the mechanism of PsaA-facilitated metal binding. However, the conformational dynamics of metal-free PsaA in solution remain unknown. Here, we use continuous wave electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations to study the relative flexibility of the structural domains in metal-free PsaA and its distribution of conformations in solution. The results show that the crystal structure of the metal-free PsaA is a good representation of the dominant conformation in solution, but the protein also samples structurally distinct conformations that are not captured by the crystal structure. Further, these results suggest that the metal binding site is larger and more solvent exposed than indicated by the metal-free crystal structure. Collectively, this study provides atomic-resolution insight into the conformational dynamics of PsaA prior to metal binding and lays the groundwork for future EPR and MD based studies of PsaA in solution

Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance.

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer's and Huntington's disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, "On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you.